This invention relates to a process of preparing purified aqueous indole solution. More particularly, this invention relates to a process of preparing substantially purified aqueous indole solution from crude indole containing an organic impurities.
A number of methods are known for synthesizing useful compounds by an enzyme reaction employing indole as substrate of the enzyme. A typical example of such a method is a process of synthesizing L-tryptophan from indole and L-serine using tryptophan synthase, and this process is now industrially practiced.
In general, indole is produced by a chemical synthesis process or by fractional distillation of coal tar, and indole produced by such a process usually contains substituted indoles such as ethylindole and other aromatic compounds as impurities. Some of these impurities inhibit or even completely deactivate the enzyme if they exist in the reaction mixture. Therefore, in order to conduct the enzyme reaction fluently, these impurities should not exist in the reaction mixture and should not be accumulated in the reaction mixture.
On the other hand, for most of the enzymes which are used in enzyme reactions, water acts as an ideal solvent. Therefore, in most cases, the enzyme reaction is conducted in aqueous solution. Further, since most enzymes exhibit high activity at a temperature of 15.degree. C. to 50.degree. C., enzyme reactions are usually conducted in this temperature range.
However, indole is insoluble in water and its solubility in water at room temperature is only 3-4 g/l. Therefore, in an enzyme reaction in which indole is used as a starting material, it is not easy to prepare and supply aqueous indole solution, especially quantitatively.
Conventional methods of preparing aqueous indole solution include a method in which solid indole powder is dissolved in water or in aqueous solution which already contains indole by vigorous agitation to prepare aqueous indole solution, and the undissolved indole is recovered by separating solid from liquid, and a method in which indole solution is prepared by dissolving indole in a solvent which is miscible with water such as lower aliphatic alcohol, e.g., methanol, and the indole solution is then mixed with aqueous indole solution.
By these methods, the quantitativeness of the indole concentration in the aqueous solution is assured However, when industrially produced crude indole is used, the impurities contained therein are also dissolved in the aqueous solution and are supplied to the enzyme reaction. Thus, when crude indole is used in the above-mentioned conventional methods, purification of the indole is not performed, so that it is not preferred for use in the enzyme reaction. Further, in cases where a solvent miscible with water is used, the solvent per se denatures, inhibits or deactivates the enzyme, so that the use of such a solvent should be avoided.
As an enzyme reaction in which indole is a substrate, a method using an organic solvent in the reaction has been proposed.
That is, Japanese Patent Publication No. 45593/81 discloses to conduct an enzyme reaction in which the substrate of the enzyme is almost insoluble in the enzyme-containing solution, in the presence of an organic solvent which is immiscible with water but miscible with the substrate. However, in this method, the function of the organic solvent is to assure the substrate in substantially the same concentration as the saturated concentration thereof in the aqueous phase in which the reaction by enzyme is conducted.
Japanese Patent Disclosure (Kokai) No. 11187/84 discloses that in an enzyme reaction in which at least one substrate inhibits the enzyme activity, an organic solvent which is immiscible with water but miscible with the substrate is added so as to reduce the substrate concentration in the aqueous phase to a level at which the enzyme activity is not inhibited. In this reference, a method of enzymatically synthesize L-tryptophan using indole as a substrate is disclosed.
Further, Bang et al disclose a method of synthesizing L-tryptophan from indole and L- or DL-serine using E. coli cells, in which indole is dissolved in an organic solvent immiscible with water and the indole solution is used as a source of indole in the enzyme reaction. (Biotechnology and Bioengineering. vol. XXV 999-1011, 1983).
These methods in which an organic solvent which is immiscible with water is used are effective methods in the case where a substance such as indole which is substantially insoluble in water is a substrate of an enzyme. However, these methods do not care at all about the impurities contaminated in indole, and they intend to employ pure indole. Therefore, in these methods, the role of the organic solvent is only to restore indole and supply it to the aqueous phase at a fixed concentration when conducting an enzyme reaction.
However, in cases where the indole which is used as a starting material is crude indole containing the organic impurities, care should be taken for the selection of the organic solvent. The present inventors found that o-ethylaniline or 3-methylindole which may be contained in the crude indole inhibits the enzyme, tryptophan synthase which synthesize tryptophan from indole and L-serine at a level of about 200 ppm, and 2-ethylindole inhibits the enzyme at a level of as low as about 20 ppm. There are some other impurities which inhibit the enzyme if they exist at a high level of 1000 ppm or more.
Thus, even if the organic solvent which is immiscible with water restores indole and assures to supply indole in the aqueous phase at a constant concentration, if the organic impurities are distributed to the aqueous phase to the same degree as indole, the impurities co-exist in the enzyme reaction system to inhibit the enzyme. Further, even if an organic solvent of which distribution ratio of the impurities is somewhat lower than that of indole is employed, since the substrate indole is consumed by the enzyme reaction while the impurities are usually not consumed in the reaction, the impurities accumulate in the reaction system. Thus, if an organic solvent with a distribution ratio of the impurities to the aqueous phase of not sufficiently small is employed, it is not easy to prevent the inhibition of the enzyme reaction or the contamination of the product of the enzyme reaction, so that it is difficult to conduct the enzyme reaction fluently.
However, no finding or consideration is disclosed in the prior art in which the organic solvent which is immiscible with water is employed. As a countermeasure for solving the problem of the contaminating impurities, to purify the crude indole is considered. However, although indole may be purified by rectification or recrystallization, most of the contained impurities are substituted derivatives of indole with physiological properties similar to those of indole, so that the purity of indole which may be attained by the purification operation such as rectification is limited. Among the purification operations, recrystallization is a suitable method for obtaining indole with high purity. However, the recovery of indole is low, and the operation has a problem in the accumulation of the impurities in the filtrate Further, the cost for conducting recrystallization is high. Thus, for industrial production, a process of purification without recrystallization is demanded.